Use of oxalic acid for the hydrolysis of steroid conjugates in pregnancy analysis

ABSTRACT

A method for the determination of pregnancy estrogen concentrations by hydrolysis and/or cleavage of conjugated steroids found in urine by treatment with organic acids. The urine of a pregnant woman contains &#39;&#39;&#39;&#39;placental estriols&#39;&#39;&#39;&#39; that may be hydrolyzed with oxalic acid to liberate estrogens. The liberated estrogens are extracted into an organic solvent, which may be compared with standard concentrations of estriol by known colorimetric methods.

i United States Patent [111 an 1] w [72] Inventors Paige K. Besch [56]References Cited N Pi r g C l b Oh UNITED STATES PATENTS lc Gas orys cuml0 1 A 2 [2}] Appl No. 827,955 3,520,658 7/ 970 nyanwu 3/230 B [22]Filed May 26, 1969 Primary Examiner-Morris O. Wolk [45] Patented Oct.26, 1971 Assistant Examiner-R. M. Reese [73] Assignee Searle ReferenceLaboratories, Inc.

[54] USE OF OXALIC ACID FOR THE I-IYDROLYSIS OF STEROID CONJUGATES INPREGNANCY Attorney-Fay, Sharpe and Mulholland ABSTRACT: A method for thedetermination of pregnancy estrogen concentrations by hydrolysis and/orcleavage of conjugated steroids found in urine by treatment with organicacidsv The urine of a pregnant woman contains placental estriols" thatmay be hydrolyzed with oxalic acid to liberate estrogens. The liberatedestrogens are extracted into an organic solvent, which may be comparedwith standard concentrations of estriol by known colorimetric methods.

USE OF OXALIC ACID FOR THE IIYDROLYSIS OF STEROID CONJUGATES INPREGNANCY ANALYSIS BACKGROUND OF THE INVENTION The sterols comprise avast group of naturally occurring substances which are derivatives of aparent hydrocarbon, cyclopentanoperhydrophenanthrenezcyclopentanoperhydrophenanthrene cholesterol Compounds which arechemically related to cholesterol are designated steroids according toR. K. Callow and F. G. 40

Young, Proc. Roy. Soc. 157 A, 194 (1936). Four specific examples ofcompounds of steroid nature are:

Estradlol-Ufl Progesterone CH CH3 Androstane Testosterone LII Thechemistry of the steroids is well known and they play an important rollin life processes. General information concerning steroids is availablein texts such as G. L. Jenkins W. H. Hartung, K. E. Hanlin, Jr. and J.B. Data, Chemistry ofOrganic Medicinal Products (4th ed. 1957); and .l.S. Fruton, S. Simmonds, General Biochemistry (2d ed. 1961 The steroidhormones are classified into several groups (estrogens, androgens, etc.)depending on their physiological effects which, in turn, depend on theirchemical structure. In mammals, steroids are produced by the adrenals,gonads, placenta and fetal tissues. Steroids appear in the urine as thefree steroid or in a conjugated (combined) form. The conjugated formresults from the interaction of free steroids with a conjugating agent.Some chemical agents capable of conjugation with the steroid arephosphoric sulfuric and glucuronic acids.

Most of the steroids in the urine are present in the form of aconjugated derivative which is not extractable with common organicchemical solvents. Certain methods for the determination of steroidconcentrations involve, necessarily, a release of the steroid from itsconjugating agent by hydrolysis (i.e., cleavage of the bond between thesteroid and its conjugating agent). This hydrolysis step cleaves thebond between the conjugating agent and steroid and, therefore, releasesthe free ste roid. The free steroid is soluble in certain organicsolvents such as ether, ethyl acetate and chloroform. Thus, thehydrolysis procedure serves to free the steroid and allows it to beextracted from the aqueous (urine) sample into the selected organicsolvent. Hence, it can be removed from the biological fluid.

Hydrolysis has, in the prior art, been accomplished by the use ofthecombination of strong mineral acids such as sulfuric or hydrochloricacids and heat. Inherent in hydrolysis by the use of strong mineralacids is the danger of chemical transformations to the steroid which mayproduce a derivative incapable of responding to the analytical method.

Attempts at using less severe hydrolytic procedures, such as solvolyticor enzymatic methods, have the disadvantage of extensive timerequirements for analysis. The use of an enzymatic method alsointroduces a cost disadvantage. Addi tionally, in commercial steroidanalysis kits which can be shipped to hospital or private laboratories,the shipping restrictions on the strong mineral acids used for thehydrolysis step and the danger of spillage during shipping presentadditional undesirable factors.

This invention proposes a new and unique method of hydrolyzing thesteroid without the use of strong mineral acids involving the use of thefairly strong crystalline dicarboxylic acid, oxalic acid (pK,=l.42, pK=4.3l Advantages of the use of oxalic acid for the purpose ofhydrolyzing the steroid over strong mineral acids are:

l The Absence of Artifact Formation For example, reactions of thefollowing nature are HCl In this example the formation ofa 3,B-chloroanalog is possible if hydrochloric acid is used for the hydrolysis step.

2. The Absence of Undesirablc Chemical Transformations I GrossDegradation of the Steroid It is possible that the combination of heatand mineral acids could cause the destruction of the steroid nucleus.

3. Speed of Analysis Approximately four hours is required for adetermination. Enzymatic or solvolytic methods for hydrolysis wouldrequire longer periods of time for analysis.

4. Shipping Convenience Shipping restrictions applicable to sulfuric andhydrochloric acids would not be applicable to oxalic acid.

5. Shipping Safety In the event of damage to the shipped steroidanalysis kit, personnel would be exposed to a relatively nontoxic powderinstead of a dangerous mineral acid.

6. Analytical Convenience An analytical laboratory could obviate theneed for a fume hood if the practically nontoxic oxalic acid were used.In addition, the personnel would not have to be exposed to the dangerousmineral acid fumes. The method of hydrolysis using oxalic acid is veryaccurate, inexpensive, and faster than safe enzymatic or solvolyticmethods.

SUMMARY OF THE INVENTION This invention, then, proposes the use ofoxalic acid as a means of hydrolyzing conjugated steroids to give a freesteroid which can be extracted from the biological fluid and analyzed bya suitable chemical method.

PREFERRED EMBODIMENT It has been shown that during pregnancy, while allestrogens rise somewhat, there is an overwhelming increase in estriol,particularly during the third trimester, which exceeds any quantity thatcould be attributed to maternal secretion. Recent studies have shownthat the large quantity of estriol excreted in the maternal urine is aresult of the placental conversion to estriol of l6-OHdehydroepiandrosterone, which is secreted by the fetal adrenal. It isthis phenomenon which makes the detennination of placental estriol" oneof the best indices of the well-being of the fetal-placental complex.

Although methods are available which are specific for estriol, it ismore convenient to measure the total estrogens. The estriol-specificmethods are more complicated and lengthy than the total estrogentechnique. Since rapid return of the data to the clinician is frequentlyimperative, this invention is designed to measure total estrogens in arelatively short period of time.

Urinary estrogens are consistently decreased in pregnancies complicatedwith maternal diabetes, toxemia of pregnancy, and premature placentalsenescence. New developments in extraction of the Kober chromogens fromthe nonspecific chromogens, which previously have plagued the analyst,have made it possible to detect quantities heretofore impossible insmall amounts of urine.

In general a description of the instant invention is as follows. Amilliliter aliquot of a 24 hour urine specimen containing a steroidconjugate is hydrolyzed optionally in the presence of a salt withcrystalline oxalic acid which dissolves upon heating, and recrystallizesagain upon cooling. The liberated estrogens (estriol comprising over 95percent of the total) are extracted into a solvent, an aliquot of whichis transferred to a color tube and dried. Known quantities of estriolare pipetted into identical tubes and the color developed, extracted,and compared in a spectrophotometer at various wavelengths, making useof the Allen Correction to minimize the background effect.

One or more of the following steroid conjugates, which may be present inthe urine of a pregnant woman can be hydrolyzed with oxalic acid toyield the beneficial results of the invention:

STEROID CONJUGATES Estra-l ,3,5( l)-trienl 7-one-3-yl sulfate Estra-l,3,5( l0)-trien-l7 [3-01-31 sulfate Estra-l ,3,5( l0)-trien-3,l 7B-diylsulfate Estra-l ,3 ,5( l0)-trien- 1 6a,! 7B-diol-3-yl sulfate Estra-l,3,5( l 0)-trien-l7-one-3-ylB-D-glucopyrahosiduronicD-glucopyranosiduronic acid Q Estra-l ,3,5( l0)-trien-3, l7fi-diol'a-ylB-D-glucopyranosiduronic acid Estra-l ,3 ,5( l0)-trien-3, l6a-dioll 7fl-ylB-D-glucopyranosiduronic acid Estra l ,3 ,5( l0)-trienl7B-ol-3 l 6a-diylB-D-glucopyranosiduronic acid Estra-l ,3 ,5( l0)-trienl6a-ol-3-yl sulfate- 1 7B-yl/3-D-glucopyranosiduronic acid Estral ,3 ,5(l0)-trienl 7B-ol-3, l 6a-diyl sulfate Androstan-S -en- 1 7-one-3B-ylsulfate SB-Androstanl 7-one-3B-yl sulfate 5a-Androstan-l7-one-3a-ylsulfate Androstan-S -en- 1 7-one-3B-ylB-D-glucopyranosidurmic acidSB-Androstanl 7-one-3a-ylB-D-glucopyranosidurmic acid SIS-Androstanl7-one-3aylB-D-glucopyranosiduramic acid Androstan-4en-3-onel7B-ylfi-D-glucopyranosidunnic acid I 7012 l -dihyrdroxy-5B-pregnan-l l,20-dione-3a-ylBB-D- gluocopyranosidurmic acid 1 1B, 1 701,21-trihydroxy-5B-pregnan-20-one-3a-ylB-D-glucopyranosidurmic acid 301, I75, l 7a-trihydroxy-SB-pregnan-ZO -one-2 l -ylB-D-glucopyranosidurmicacid In each case hydrolysis with oxalic acid provides a free steroid orestrogen capable of colorimetric analysis by the measurement of totalestrogen concentration.

APPLICATION ON PREGNANCY DETERMINATIONS The reaction between theaforesaid steroids contained in the urine of a pregnant woman withoxalic acid according to the method of this invention should beconducted at the boiling point of the hydrolysis solution. In the testprocedure outlined, the hydrolysis solution will contain gram of oxalicacid and 200 milligrams of sodium chloride. The boiling temperature inmost cases, depending upon atmospheric pressure and other variables,will be between centrigrade and l l0 centigrade. The most preferredtemperature range being approximately to centigrade.

The ratio of dry, crystalline oxalic acid to other satisfactory acid tourine sample will vary from approximately l/2 gm. per ml. to 2 gm. perml. The time for heating the acid and conjugated steroid hydrolysissample in order to complete the hydrolysis reaction will usually varybetween 20 minutes and minutes. The following is an illustration of astep-by-step technique for using the invention to determine estrogenconcentrations in urine:

EXTRACTION 1. Add Urine. To hydrolysis tube containing 200 mg. NAC] and1 gm. oxalic acid, add 1.0 ml urine, cap, punch pinhole in cap.

2. Boil 1 hour. Mix several times during the first few minutes todissolve acid crystals.

3. Cool SLOWLY to room temperature so that large crystals form. (Inclinetube so that crystals form on the side of the tube instead of thebottom.)

4. Add Extraction Solvent (dichloromethane). Add 10.0 ml of theExtraction Solvent to the cooled tube.

5. Cap again, place finger over pinhole and shake vigorously for 30seconds.

6. Spin in centrifuge until solvent is clear.

7. Aspirate urine from solvent.

8. Transfer 5.0 ml of solvent from the specimen tube to a color tube.

9. Pipette 0.5 and L0 ml STANDARD SOLUTION (10 a g./ml. of estriol) into2 other color tubes.

10. Dry the 3 tubes. Evaporate the 3 color tubes to dryness.

COLOR DEVELOPMENT 1. Add Reagent. To each color tube, add l.5 ml. ofCOLOR REAGENT (sulfuric acid containing hydroquinone). Mix.

2. Boil 20 minutes, mixing twice again during the first 6 minutes.

3. Cool in a cold water bath.

4. Add water, 0.5 ml. distilled water to each tube. Mix.

5. Replace in boiling bath for 10 minutes.

6. Chill in ice bath.

COLOR EXTRACTION I OH mg. of pure solid estriol is contained and mixedwith 20 ml. of pure ethyl alcohol.

Thereafter, color development and color extractions are run so that thestandard estriol solutions may be compared to the specimen of estriolthat is yielded from the urine sample. The spectrophotometric readingsare taken at 532-534 mm, and at 504 mm. and 564 mu on aHitachi-Perkin-Elmer spectrophotometer. The 504 and 564 readings aretaken, averaged and subtracted from the 532-5 34 peak reading.

The Kober method of colorimetric analysis is well known and the lttn'chmodification was published in I959.

Calculations for 24 hour specimen as to actual concentration of estriolin the specimen urine may be calculated according to the following:

4 mu. l A554, mu.

A5 4 mu. o

+glucuronic acid (17-13 Estradlol-glucuronldc) (17-13Estradiol-B-sulphate) EXAMPLE One ml. of a 24 hour urine sample ofafemale human who is known to be in the third trimester of pregnancy isadded to a test tube containing one gram of oxalic acid and 200milligrams of sodium chloride. The contents of this test tube are boiledfor 1 hour at 100 Centigrade. During this boiling, of the sample, thecontents of the tube are mixed several times during the first fewminutes to dissolve the acid crystals. Subsequently, to the l-hourboiling time, the sample is cooled slowly to room temperature so thatlarge crystals form on the side of the tube rather than the bottom ofthe tube. Thereafter, 10 ml. ofdichloromethane is added.

For the extraction solvent dichloromethanemay be used or some equivalentsolvent such as chloroform, diethyl ether or ethyl acetate.

The sample is mixed thoroughly with the extraction solvent by shakingvigorously for 30 seconds. The sample is then spun in a centrifuge untilthe solvent is clear. The urine is then aspirated from the solvent.

At this point 5 ml. of specimen solvent is transferred from the specimentube to a color tube. For purposes of comparison, 0.5 and 1.0 miistandard solution of estriol are added to two other color test tubes.The concentration of the standard estriol solution should beapproximately l0 mg./ml. In the preferred method of making up a standardsolution, 200

2 CA unk. C oncentra t aion unk.( u M CA std. Concentration std.

ug./tubeX2XT.V.

3. M1000 rng./T.V.

Where A. Absorbance mg. e milligram CA Corrected Absorbanco unk. unknownm t= millimicrons std. standard g. microgram T.V. Total 24 hour volumein ml.

In this example the mg./T.V. was 14.4, which is an average estriolconcentration for a woman in the third trimester.

CALCULATlONS FOR SINGLE VOIDED SPECIMENS The laboratory which is set upto determine creatinine in urine can speed the data to the physician byperforming both an estriol and a creatininc determination on a singlevoiding of urine and expressing the result as an estrogen/creatinineratio (Dickey et al., Am. J. Obst. & Gynec., 94, 59l, 1966).

l Calculate mg. estrogens as above and express as mg./T.V.

2. Measure and calculate creatinine in specimen and expressasgramsl'l.V.

3 Ratio EIC= mg. estrogen/TN. grams creatinine/T.V.

' reaching a level of 8 mg. or higher as the third trimester is entered,rapidly rising to levels as high as 50 .mg./24 hours at I term. Thediagnostic significance of the hydrolysis reaction of this inventionlies in its relationship to the status of the developing feta-placentalunit. A value which drops progressively during the third trimesterpredicts impending fetal distress and may indicate that an earlydelivery by caesarean section is necessary to prevent fetal demise. Thelaboratory should note carefully all values on a given patient whichdecline steadily, especially when the values fall below 12 mg./24 hours,reporting these values to the physician at once.

It will also be understood that the foregoing description is merelyillustrative of the invention, and that various changes in thetechniques, conditions, proportions, compositions, and other factors setforth may be made without departing from the spirit of the invention asdefined in the appended claims.

The claims:

1. The method of determining the concentration of total estrogenspresent in the urine of a pregnant female, which comprises:

a. mixing the females urine that contains conjugated estrogens withoxalic acid to form a hydrolysis sample;

b. boiling the hydrolysis sample for a period of time suffrcient tohydrolyze all estrogens present;

c. separating the urine from the estrogen compounds and d. quantitivelydetermining the concentration of estrogens present.

2. The method of claim 1 wherein the estrogens present are conjugates ofan estrogen and an acid selected from the group consisting ofphosphoric, sulfuric and glucuronic acids.

3. The method of claim 1 wherein the boiling time varies ii. from 20minutes to 120 minutes.

4. The method of claim 1 wherein the ratio of dry, crystalline oxalicacid to urine sample varies from approximately 1/2 gm. per ml. to 2 gm.per ml.

5. The method of claim 1 wherein a solvent for the estrogens is admixedwith the sample after boiling.

6. The method of claim 5 wherein the solvent is selected from the groupconsisting of dichloromethane, chloroform, diethyl ether and ethylacetate.

7. The method of claim 1 wherein boiling of the hydrolysis sample isconducted at a temperature range of -l 10 C.

8. The method of claim 1 wherein hydrolysis takes place in the presenceof a salt.

9. The method of claim 8 wherein the salt is sodium chloride.

10. The method of making a colorimetric determination of totalhydrolyzed steroids in the urine of a pregnant female comprising thesteps of:

a. collecting a 24-hour urine specimen;

b. adding a 1 ml. aliquot of said specimen to 200 mg. of

' sodium chloride and 1 gram of oxalic acid to form a hydrolysis sample;

c. boiling the hydrolysis sample for a period of time adequate tohydrolyze all conjugated steroids contained in the urine specimen;

. adding a solvent to extract the hydrolyzed steroids;

e. mixing the extraction solvent and hydrolyzed steroid sample;

. separating urine from the solvent containing dissolved hydrolyzedsteroids;

making up a standard solution of estriol in a solvent and adding colordevelopments fluids thereto; h. making a colorimetric comparison betweenthe known concentration of estriol and the hydrolyzed steroids insolution.

11. The method of hydrolyzing a steroid conjugate comprising:

a. mixing a steroid conjugate in aqueous solution with oxalic acid;

b. boiling the mixture for a period of time sufiicient to hydrolyze thesteroid conjugate.

PAGE 1 OF 3 UNITED STATES PATENT OFFICE PO-105O (5/69) CERTIFICATE OFCORRECTION Patent No. 3, 6 5, 9 Dated October 26, 97

e tofls) Paige K. Besch and Nicholas Vorys It is certified that errorand that said Letters Patent are appears in the above-identified patenthereby corrected as shown below:

Column 5, line 75, "-31" should be Column 4, line 3, "-ylB" should be ylB Column 4, line 3, '--glucopyrahosiduromic" should beglucopyranosiduronic Column L, line L, delete "D-glucopyrano=siduronic."

Column L, line 5, "diol-a-ylB" should be --diol-l6a-yl 6.

Column 4, line 7, "ylB" should be yl B Column 4, line 9, "diylB" shouldbe yl B Column line 11, "5 16" should be yl Column l, line 15, delete.

Column 4, line 17, "ylB" should be yl B Column 4, line 18, "ylfi" shouldbe yl B PAGE 2 OF 5 POMS) UNITED STATES PATENT OFFICE (s/s r JCERTIFICATE OF CORRECTION Patent No. 3, -5, 9 Dated October 26, 1971Invent Paige K. Besch and Nicholas Vorys It is certified that errorappears in the above-identified patent and that said Letters Patent arehereby corrected as shown below:

Column 4, line 19, "56" should be 5d Column line l9, "ylB" should be ylB Column t, line 20, "ylB" should be yl B Column l, line 21,"l7a2l-dihyrdroxy" should be 17a El-dihydroxy Column t, line 22,"ylBB-D-" should be yl 6-D Column l, line 23, "ylB" should be yl BColumn 4, line 19, "glucopyranosiduramic" should be glucopyranosidurmicColumn t, line 5, "to" should be or Column 5, line 16, "mu" should be mColumn 5, line 7 "mg" should be g Column 6, line 1 "mg" should be gColumn 6, line 6, "mm should be m Column 6, line 7, "50 mm. and 56 k mu"should be-50h m and So l m Column 6, line 17, 3 locations "mu" shouldPAGE 5 OF 5 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION PatentNo. 5 615,229 Dated October 26, 1971 Inventor) Paige K. Bosch andNicholas Vorys It is certified that error appears in theabove-identified patent and that said Letters Patent are herebycorrected as shown below:

Column 6, line 50, "ug" should be g Column 6, line 5 "ug" should be gSigned and sealed this 18th day of July' 1972.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. ROBERT GOTTSCHALK Attesting Officer Cormnissionerof Patents

2. The method of claim 1 wherein the estrogens present are conjugates ofan estrogen and an acid selected from the group consisting ofphosphoric, sulfuric and glucuronic acids.
 3. The method of claim 1wherein the boiling time varies from 20 minutes to 120 minutes.
 4. Themethod of claim 1 wherein the ratio of dry, crystalline oxalic acid tourine sample varies from approximately 1/2 gm. per m1. to 2 gm. per m1.5. The method of claim 1 wherein a solvent for the estrogens is admixedwith the sample after boiling.
 6. The method of claim 5 wherein thesolvent is selected from the group consisting of dichloromethane,chloroform, diethyl ether and ethyl acetate.
 7. The method of claim 1wherein boiling of the hydrolysis sample is conducted at a temperaturerange of 90*-110* C.
 8. The method of claim 1 wherein hydrolysis takesplace in the presence of a salt.
 9. The method of claim 8 wherein thesalt is sodium chloride.
 10. The method of making a colorimetricdetermination of total hydrolyzed steroids in the urine of a pregnantfemale comprising the steps of: a. collecting a 24-hour urine specimen;b. adding a 1 m1. aliquot of said specimen to 200 mg. of sodium chlorideand 1 gram of oxalic acid to form a hydrolysis sample; c. boiling thehydrolysis sample for a period of time adequate to hydrolyze allconjugated steroids contained in the urine specimen; d. adding a solventto extract the hydrolyzed steroids; e. mixing the extraction solvent andhydrolyzed steroid sample; f. separating urine from the solventcontaining dissolved hydrolyzed steroids; g. making up a standardsolution of estriol in a solvent and adding color developments fluidsthereto; h. making a colorimetric comparison between the knownconcentration of estriol and the hydrolyzed steroids in solution. 11.The method of hydrolyzing a steroid conjugate comprising: a. mixing asteroid conjugate in aqueous solution with oxalic acid; b. boiling themixture for a period of time sufficient to hydrolyze the steroidconjugate.